This invention relates to the solubilization and stabilization of compounds using cyclodextrins.
Cyclodextrins are a group of compounds consisting of, or derived from, the three parent cyclodextrinsxe2x80x94alpha-, beta- and gamma-cyclodextrins. Cyclodextrins have numerous uses based on their ability to solubilize and complex chemicals.
Alpha-, beta- and gamma-cyclodextrins cyclodextrins are simple oligosaccharides consisting of six, seven or eight glucose residues, respectively, connected to macrocyles by alpha (1 to 4) glycosidic bonds. Each of the glucose residues of a cyclodextrin contains one primary (O6) and two secondary hydroxyls (O2 and O3) which can be substituted, for example, methylated. Many cyclodextrin preparations in practical use are mixtures of chemically individual derivatives in which only a part of hydroxyl groups were substituted and which differ in number and position of these substituents.
This invention uses many different cyclodextrin derivatives including several mixtures of partially methylated cyclodextrins. One composition is a commercial preparation (Wacker Chemie, Beta W7M1.8) in which the methyl substituents are about equally distributed between the primary and secondary hydroxyls of glucose residues; it is abbreviated here as RAMEB. A second class has methyls predominantly substituting for hydrogen at the secondary hydroxyls. These are prepared as described in U.S. Pat. No. 5,681,828 and are referred to as Pitha""s methyl derivatives. A third type of methylated cyclodextrins is formed by those cyclodextrin derivatives or their mixtures that have more than half of their secondary hydroxyl groups (i.e., O2 and O3) methylated. For brevity, these are called xe2x80x9chighly methylated cyclodextrins.xe2x80x9d Other mixtures of cyclodextrin derivatives used in this work are partial 2-hydroxypropyl ethers, abbreviated as HPACD, HPBCD or HPGCD for derivatives of alpha-, beta- and gamma-cyclodextrins, respectively.
In addition to the mixtures described in the preceding paragraph, the invention also uses five chemically individual cyclodextrin derivatives: (1) fully methylated alpha-, beta- and gamma-cyclodextrins, abbreviated as TRIMEA, TRIMEB and TRIMEG, respectively, (2) dimethyl derivative of beta-cyclodextrin, DIMEB, in which all glucose residues carry methyl groups on O2 and O6, and (3) maltosyl derivative of beta-cyclodextrin, G2, in which beta-cyclodextrin carries maltosyl residue on O6. The compounds under (1) are of the group of highly methylated cyclodextrins and, like the highly methylated mixtures, have unique properties. Compounds under (2) and (3) do not belong to this group.
Cyclodextrins solubilize insoluble compounds into polar media by forming what is known as an inclusion complex between the cyclodextrin and the insoluble compound; cyclodextrin solubilization power is directly proportional to the stability of the complex. Inclusion complexes are non-covalent associations of molecules in which a molecule of one compound, called the host, has a cavity in which a molecule of another compound, called a guest is included. Derivatives of cyclodextrins are used as the hosts and the insoluble compound is the guest.
Although cyclodextrins and derivatives solubilize many compounds, they are not useful in all cases. For numerous compounds of general interest, cyclodextrins do not have sufficient solubilizing power to make their use practicable. Overcoming this defect requires using large amounts of host compound. However, this is not only uneconomical (making cyclodextrins too expensive for many applications), but also dangerous. Cyclodextrins in very large amounts can boost the effects of various toxicants potentially present outside and in the body itself (Horsky, J. and Pitha, J., J. Incl. Phen. and Mol. Rec. Chem., 18, 291-300, 1994).
Previous art relevant to the current invention discusses three areas of interest: a) highly methylated cyclodextrins, b) doubling cavity size by association of two cyclodextrin moieties and c) forming salts of guest compounds and choosing counterions when forming complexes between cyclodextrin hosts and ionic guests.
Previous art concerning modification of cyclodextrin hosts has been marginally concerned with highly methylated cyclodextrins. The reason for this lack of interest is that the most accessible compound of this group, TRIMEB, was found to be a weak host for several guests (J. Szejtli, Ccylodextrin Technology, Kluwer Academic Publishers, Dordrecht, 1988, p.56). TRIMEA, since it is a derivative of the smallest parent cyclodextrin, was assumed to have a too small cavity. See, for example, A. R. Hedges, Chem. Rev., 98, 2035-2044, 1998; K. Uekama et al., Chem. Rev., 98, 2045-2076, 1998; U.S. Pat. No. 4,687,738; Japanese Patent JP 10319587A; J. Szejtli, Cyclodextrin Technology, Kluwer Academic Publishers, Dordrecht, 1988 and references therein.
Previous art concerning doubling cavity size by using two cyclodextrin moieties relied on connection of these moieties by chemical (covalent) bonds. Specific chemical connection is required for such designs to be effective (K. Fujita et al., J. Chem. Soc. Chem. Commun., 1277-1278, 1984; R. Breslow et al., J. Amer. Chem. Soc., 118, 8495-8496, 1996). Additionally, molecules of some guests are known to form complexes with two molecules of the same cyclodextrin derivative. Such complexes are termed 1:2 complexes and often accompany the usual 1:1 complexes. The present invention shows that some combinations of two different cyclodextrin derivatives lead to better inclusion by host molecules.
Previous art describing complex formation by ionic guests is extensive. A review of those systems, in which parent cyclodextrins were used, counts 271 systems for alpha-cyclodextrin and 342 for beta-cyclodextrin alone (K. A. Connors, J. Pharm. Sci., 84, 843-848, 1995). Statistically, the complexes of anionic guests have about twice the stability compared to corresponding uncharged guests, but there are numerous cases where the situation is reversed and the uncharged guest is preferred. It is not possible to predict whether a specific guest will have greater stability in charged or uncharged state. In previous art, the bases used to form anions of guests were non-volatile and inorganic; many of the guests described there have been used as drugs.
U.S. Pat. No. 4,727,064 discloses that ionization of guest molecules may be an important factor in formation of inclusion complexes of hydroxypropyl derivatives of cyclodextrins and that formation is affected by counterions. Solubilization of retinoic acid in its acidic form and in the form of its sodium, choline and ethanolamine salts were compared and found to increase in that order. The patent also teaches how to prepare such complexes in solid form.
U.S. Pat. No. 5,120,720 and an article by Pitha, Hoshino, Torres-Labandeira and Irie (International Journal of Pharmaceutics, 80, 255-258, 1992) describe a method for preparing inclusion complexes in which a volatile base, ammonia, was used to bring fast dissolution of acidic guests wherein the cyclodextrin derivative was added immediately after the dissolution. The majority of the volatile base was removed with water after solid complexes were prepared.
Related work described later taught addition of bases or organic acids facilitated the solubilization of drugs while cyclodextrins or cyclodextrin derivatives were already present in solutions (E. Fenyvesi et al., The 7th International Cyclodextrins Symposium, Tokyo 1994, Proceedings, pp. 414-418; E. Fenyvesi et al., The 8th International Cyclodextrin Symposium, Budapest 1996, Programme and Abstracts 3-p48; Italian Patent Application M193 A000 141; PCT WO 95/28965; U.S. Pat. No. 5,773,029). The inventors call their system a multiple complex formation by cyclodextrin derivatives. In their claims, they specify that the characteristic of multicomponent inclusion complexes is the simultaneous salt formation and complexation.
The present invention discloses new methods which have wide applicability to potentiate formation of inclusion complexes between cyclodextrins and guests. These methods were discovered during attempts to solubilize a test compound, S-farnesylthiosalicylic acid (abbreviated as FTS), a potential drug. Highly methylated cyclodextrins were found to form non-covalent complexes with less substituted cyclodextrins spontaneously and rapidly. Such complexes of two cyclodextrin derivatives were found to have a better ability to form an inclusion complex with specific guests than either of the two cyclodextrin derivatives alone. By this procedure, FTS in its acidic form, which is highly water insoluble, was solubilized. Salts of FTS, which have higher water solubility than the acidic form, were well solubilized by these complexes of two cyclodextrin derivatives. However, in this case, even one cyclodextrin derivative leads to useful dissolution. Complexes of two cyclodextrin derivatives, or their components also dissolved representative organics and stabilized colloidal inorganics. In addition to solubilizing water insoluble compounds, complexes of two cyclodextrin derivatives have applications in analytical chemistry for separation of various compounds based on their ability to form inclusion complexes with two cyclodextrins.
In order to evaluate systematically the solubilization potency of the available cyclodextrin preparations and of their combinations, test compound, S-farnesylthiosalicylic acid (abbreviated as FTS), was used. This compound is a candidate anticancer drug and, in its acidic form, has very low water solubility. Several combinations of cyclodextrin preparations were found to be better solubilizers of FTS in acidic form than any cyclodextrin preparation alone.
Example 1 shows potentiation of inclusion complex formation using various combinations of two cyclodextrin hosts. The best combinations are those containing highly methylated alpha-cyclodextrin and less methylated, or substituted, beta-cyclodextrin derivatives. The latter may lack any substitutions at the secondary hydroxyls (e.g., G2). The highly methylated alpha-cyclodextrin can be replaced by the corresponding beta- or gamma-derivatives, but the more preferred complex is formed using the former. On the basis of steric considerations, complexes also can be expected to form between highly methylated and less highly methylated alpha-cyclodextrins.
Both the type of cyclodextrin and the kind of substituents are important. The combination of alpha- and beta-cyclodextrin, when used in their hydroxypropylated form, lacks some desirable properties sought. For an improved combination, the inclusion complexes are best formed in a cooperative manner. This cooperativity is measured, in Example 1, by a cooperativity index, which denotes the solubilization of a combination divided by the sum of solubilization components acting separately. Salts of FTS have higher water solubility than FTS in the acidic form and, as shown in Example 1, easily form complexes with cyclodextrin derivatives. Thus, the present invention discloses as preferred inclusion complexes formed by two different cyclodextrin derivatives.
Example 2 shows that the cyclodextrin components of one of the cooperative combinations, TRIMEA-DIMEB, associate spontaneously by themselves; that is, the presence of a guest is not required. Example 3 shows results of evaluation by the continuous variation method indicating that, when combining TRIMEA-DIMEB, about equal amounts of these cyclodextrin components are required for optimal solubilization. Since their molecular weights are similar (1225 and 1331 respectively), this shows that the inclusion complex involves one molecule of TRIMEA and one of DIMEB. [It may be noted that a process similar to formation of inclusion complexes is used in all known living systems for repair of proteins that are in non-native conformation. One of these systems consists of a large subunit chaperonin, GroEL, which has a seven sided cavity that is capped, as by a lid, by co-chaperonin GroES (M. Shtilerman et al., Science, 284, 822-825, 1999).] The TRIMEA-DIMEB combination of this invention has a formal similarity to this chaperonin system. Further, the data in Example 3 shows that the relation between solubilization and concentration of cyclodextrin hosts is about linear. In other words, solutions of the guest in this particular combination of cyclodextrin derivatives will not precipitate the guest upon dilution with water or aqueous solvent. This is an important property for applications in pharmaceuticals and cleaning compositions. Results in Example 4 show that complexes resulting from combinations of cyclodextrins work effectively for solubilization of several water insoluble compounds in addition to FTS.
Complexes of combinations of cyclodextrins also have applications in analytical separations of compounds. Since cyclodextrins are optically active, these complexes can be used for the separation of optically isomeric compounds. A suitable system is shown in Example 5. In this system, TRIMEA-like molecules are immobilized on a solid support which is then used in a column. A mixture of guests to be separated is introduced into the column in a solution containing DIMEB-like molecules and is subsequently eluted by a similar solution. During the elution, complexes consisting of guests, DIMEB and immobilized TRIMEA form reversibly and guests are separated on basis of their ability to support formation of such complexes. The guest forming the most stable complex is eluted from the column last. Results shown later in Example 7 suggest that columns with immobilized TRIMEA also are effective for separation of proteins. In all instances of complex formation using the present invention, highly methylated alpha-cyclodextrins strongly outperformed highly methylated beta- and gamma-cyclodextrins. The use of alpha-cyclodextrins in analytical applications utilizing immobilized forms of these compounds is deemed of much value.
The highly methylated cyclodextrin component of the above complexes was, for some guest compounds, a useful solubilizing agent on its own. This is documented in the Example 6. Data shows that, for several difficult to dissolve guests, such as retinoic acid or hydrocortisone, TRIMEA outperformed other cyclodextrin hosts. This result may be explained on the basis of the recently published crystal structure of TRIMEA (T. Steiner et al., Angew. Chem. Int. Ed., 37, 3404-3407, 1998). Substitution of all secondary hydroxyls by methyls obviously leads to steric crowding which, in the case of alpha-cyclodextrin, resulted in widening the opening of the cavity on the secondary hydroxyl side and making the cavity more flat. The data in Example 6 show that this structural change makes TRIMEA a very good host on its own. Data of Example 1 shows that the same flat structure also is beneficial for formation of complexes with two cyclodextrin hosts. In these instances, the flat molecule of TRIMEA may function as a lid closing the cavity of the other cyclodextrin host. On basis of these considerations, the structural criterion for compounds of optimal activity can be definedxe2x80x94the majority of the secondary hydroxyls in these compounds must be methylated. The crowding of methyl groups starts when more than half of the secondary hydroxyls are methylated. Hence, this criterion defines compounds expected to be effectivexe2x80x94highly methylated cyclodextrins.
In Example 7, it is shown that highly methylated cyclodextrins are effective solubilizers of compounds that, because they are true macromolecules, can not be fully included. Example 8 shows that highly methylated cyclodextrins are more effective hosts than otherwise substituted cyclodextrins in formation and stabilization of inorganic guests. Colloidal particles of an electricity-conducting metal, copper, were made and stabilized; the same procedure also was used for preparing a colloidal composition of a ferromagnetic metal, cobalt. In Example 8, it also is shown that these stabilized colloidal metals are highly reactive and, thus, can be converted by chemical reaction into other colloidal compounds. Example 8, additionally, describes complexes of sulphur with highly methylated cyclodextrins; the resulting complexed sulphur has potential application in electrical batteries. Colloidal compositions of calcium fluoride, which have applications in dentistry, can be made in similar manner.
Example 9 describes preparation and subsequent solubilization of salts of FTS. The choline salt of FTS was extracted from an aqueous medium into chloroform providing a lipophilic product. The aqueous solutions of the cyclodextrin-solubilized choline salt of FTS are neutral and can be used in preparations for parenteral or sublingual administration. Ethanolamine and triethanolamine have a similar biocompactibility and structure to choline and is expected to perform similarly in the process described. The sodium salt of FTS, which was used as an intermediate in the above preparation of choline salt, also forms inclusion complexes and can be used to make pharmaceutical formulations of FTS as well. Nevertheless, all choline salts tested in Example 9 formed cyclodextrin inclusion complexes more efficiently than the corresponding sodium salts.